David D. Myrold Department of Crop and Soil Science

Oregon State University

Microbial Communities and N Cycling in Diverse Soils

Pierre Lecture 2017

N Cycle—A Historical Perspective

Alexander (1961) Introduction to Soil Microbiology p.246

Schimel & Bennett (2004) Ecology 85:591

Shift in Emphasis in N Mineralization

Daims et al. (2016) Trends Microbiol 9:699

New Processes and Organisms

N assimilation

N mineralization

N2 fixation

Denitrification

Nitrification

Anammox

Broad phylogenetic representation

Narrow phylogenetic representation

Structure-Function Connections in the N Cycle

Nitrification—A Focus of the Last Decade

• 1890—Ammonia-oxidizing bacteria (AOB) and nitrate-oxidizing bacteria (NOB) (Frankland & Frankland (1890) Phil

Trans Royal Soc B 181:107; Winogradsky (1891) Ann Inst Pasteur 5:577)

• 2005—Ammonia-oxidizing archaea (AOA) (Könneke et al.

(2005) Nature 437:543)

• 2016—Comammox, an organism (Nitrospira spp.)

capable of complete nitrification (NH4+ NO3

-) (Daims

et al. (2016) Nature 528:504; van Kessel et al. (2016) Nature 528:555)

Nitrification

Wrage et al. (2001) Soil Biol Biochem 33:1723

(amo) (hao) (nxr)

H+

Acidification Impacts of Nitrification

(1928) J Am Soc Agron 20:254

(1970) Agron J 62:106

(1977) Soil Sci Soc Am J 41:368

Nitrification Niche

The set of physical and chemical properties, as well as biological interactions, that allow the activity, growth, or survival of a particular nitrifying taxon.

Physical • Temperature

• Water

• Structure

• Porosity

• Texture

• Bulk density

Chemical • NH4

+/NH3

• pH

• C:N ratio

• Inhibitors/toxicities

• O2, CO2

• Salinity

• Micronutrients

• CEC

• Mineralogy

Niche Determining Factors

Biological • AOA and AOB

characteristics • Cell size, µmax, KS, Topt,

alternate substrates, etc.

• Ammonification rate

• Nitrate-oxidation rate

• Plant activities

Approaches for Niche Identification

• Genome analysis

• Isolate physiology

• Taxa abundance and composition (DNA)

• Activity • Transcripts (RNA)

• SIP (growth)

• Selective inhibitors

Microbial Communities of Oregon—Selection Scheme

Sampling Sites

Ammonia-oxidizer (AOA) Distribution

Burgess (unpublished data)

Ammonia-oxidizer (AOB) Distribution

Burgess (unpublished data)

AOA/AOB Ratio

Burgess (unpublished data)

Nitrite-oxidizer (NOB) Distribution

Burgess (unpublished data)

NOB:(AOA+AOB) Ratio

Burgess (unpublished data)

Taylor et al. (2013) Appl Environ Microbiol 79:6544

Differentiating AOA vs. AOB Activity

1-octyne (C8)

Acetylene (C2)

(1986) Soil Sci Soc Am J 50:1998

Lu et al. (2015) Soil Biol Biochem 85:54

Nitrification Niches Along a Forest Nitrogen Gradient

Douglas-fir Red alder

Soil Factors Related to AOA and AOB Abundance

Lu et al. (2015) Soil Biol Biochem 85:54

Soil Factors Related to AOA and AOB Abundance

Lu et al. (2015) Soil Biol Biochem 85:54

Lu et al. (2015) Soil Biol Biochem 85:54

Soil Factors Related to AOA Activity

Soil Factors Related to AOA Activity

Lu et al. (2015) Soil Biol Biochem 85:54

Nitrification Niches in Managed Soils

Response of AOA and AOB to Ammonia Addition

Giguere et al. (2015) Soil Sci Soc Am J 79:1366

Klamath Cropped (Summer)

0 20 40 60 80 100

NO

3

- + N

O2

- Accu

mu

latio

n

(mg

-N k

g-1

so

il d

-1)

0

2

4

6

8

10

12

KCl extractable NH4

+ (mg-N kg

-1 soil)

0 20 40 60 80 100 120 140 160 180

Total

AOA

AOB

Klamath non-cropped (Summer)

Ma

xim

um

Nitri

fica

tio

n R

ate

(mg

NO

3

- + N

O2

- -N

kg

-1 s

oil d

-1)

0

2

4

6

8

10

12

14

16

18

20

AOA

AOB

Non-cropped Cropped

Summer

Non-cropped Cropped

Winter

Management and Seasonal Effects on AOA and AOB

Giguere et al. (2015) Soil Sci Soc Am J 79:1366

Temperature Optima for AOA and AOB

Taylor et al. (2017) ISME J 11:896

0 10 20 30 40

Temperature (°C)

0 10 20 30 40

Nitrifica

tio

n P

ote

ntia

l

(m

ol g

-1 s

oil

d-1

)

0.0

0.1

0.2

0.3

0.4

0.5

AOA

AOB

Cropped Non-cropped

Temperature Optima for AOA and AOB

Taylor et al. (2017) ISME J 11:896

Macromolecular Rate Theory (MMRT)

Parameter Unit AOA AOB ΔHTo

‡ kJ mol-1 K-1 4.1 ± 0.7 7.2 ± 1.9 ΔCP

‡ kJ mol-1 K-1 -5.6 ± 0.6 -9.3 ± 0.9 ΔSTo

‡ kJ mol-1 K-1 -0.25 ± 0.00 -0.24 ± 0.01 Topt C 33.1 ± 1.4 20.0 ± 1.1

Ts_max C 20.9 ± 1.1 11.1 ± 0.9

Giguere et al. (2017) Soil Biol Biochem 104:30

Uncoupling of Ammonia and Nitrite Oxidation

Giguere et al. (2017) Soil Biol Biochem 104:30

Uncoupling of Ammonia and Nitrite Oxidation

Uncoupling of Ammonia and Nitrite Oxidation

Giguere et al. (2017) Soil Biol Biochem 104:30

Uncoupling of Ammonia and Nitrite Oxidation

Giguere et al. (2017) Soil Biol Biochem 104:30

Potential Mechanisms of Uncoupling/Recoupling

• Unbalanced populations—growth required

• Insufficient enzymatic capacity—protein synthesis required

• Unmatched kinetics—taxon switching

• Spatial separation

Nitrobacter nxrA

Pendleton Madras Klamath

nxr

A c

op

ies

g-1

so

il

1e+6

1e+7

1e+8

1e+9

1e+10

0 h

24 h

48 h

AOB amoA

Pendleton Madras Klamath

AO

B a

mo

A c

op

ies

g-1

so

il

1e+6

1e+7

1e+8

1e+9

1e+10

0 h

24 h

48 h

AOA amoA

Pendleton Madras Klamath

AO

A a

mo

A c

op

ies

g-1

so

il

1e+6

1e+7

1e+8

1e+9

1e+10

0 h

24 h

48 h

Nitrospira nxrB

Pendleton Madras Klamath

nxr

B c

op

ies

g-1

so

il

1e+6

1e+7

1e+8

1e+9

1e+100 h

24 h

48 h

A B

C D

AAAA

AA

AAA

AAAAA

A AAA

AA

A

AAAAA

A

AAA

AAA

AAA

Giguere et al. (submitted) Environ Microbiol

Mechanisms of Uncoupling/Recoupling

Giguere et al. (submitted) Environ Microbiol

Klamath

Time (h)9 24 48

Madras

NO3

- (-AB)

NO3

- (+AB)

Pendleton NO

2

- (-AB)

NO2

- (+AB)

*

*

*

*

*

a

b

c

A

B

C

aA

bB

cC

aAaA

bB

bB

*cC

cC

aAaA

bA

bAB

cB cC

†

†

†

†

A

B

C

0

100

200

300

400

NO

2

- or

NO

3

- (

M)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

Klamath

Time (h)9 24 48

Madras

NO3

- (-AB)

NO3

- (+AB)

Pendleton NO

2

- (-AB)

NO2

- (+AB)

*

*

*

*

*

a

b

c

A

B

C

aA

bB

cC

aAaA

bB

bB

*cC

cC

aAaA

bA

bAB

cB cC

†

†

†

†

A

B

C

0

100

200

300

400

NO

2

- or

NO

3

- (

M)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

Klamath

Time (h)9 24 48

Madras

NO3

- (-AB)

NO3

- (+AB)

Pendleton NO

2

- (-AB)

NO2

- (+AB)

*

*

*

*

*

a

b

c

A

B

C

aA

bB

cC

aAaA

bB

bB

*cC

cC

aAaA

bA

bAB

cB cC

†

†

†

†

A

B

C

0

100

200

300

400

NO

2

- or

NO

3

- (

M)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

Klamath

Time (h)9 24 48

Madras

NO3

- (-AB)

NO3

- (+AB)

Pendleton NO

2

- (-AB)

NO2

- (+AB)

*

*

*

*

*

a

b

c

A

B

C

aA

bB

cC

aAaA

bB

bB

*cC

cC

aAaA

bA

bAB

cB cC

†

†

†

†

A

B

C

0

100

200

300

400

NO

2

- or

NO

3

- (

M)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

Klamath

Time (h)9 24 48

Madras

NO3

- (-AB)

NO3

- (+AB)

Pendleton NO

2

- (-AB)

NO2

- (+AB)

*

*

*

*

*

a

b

c

A

B

C

aA

bB

cC

aAaA

bB

bB

*cC

cC

aAaA

bA

bAB

cB cC

†

†

†

†

A

B

C

0

100

200

300

400

NO

2

- or

NO

3

- (

M)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

Mechanisms of Uncoupling/Recoupling

Giguere et al. (submitted) Environ Microbiol

NO2

- (M)

0 100 200 300 400 500N

O2

- con

sum

ption

rate

(

mol g

-1 d

-1)

0.0

0.5

1.0

1.5

2.0

2.5

3.0+AB: R

2=0.65

Km=173 M NO2

-

Vmax=1.0 mol g-1

d-1

-AB: R2=0.70

Km=138 M NO2

-

Vmax=1.8 mol g-1

d-1

0 100 200 300 400 500

NO

2

- con

sum

ption

rate

(

mol g

-1 d

-1)

0

1

2

3

4

5+AB: R

2=0.75

Km=48 M NO2

-

Vmax=1.5 mol g-1

d-1

-AB: R2=0.85

Km=96 M NO2

-

Vmax=2.9 mol g-1

d-1

A

B

NO2

- (M)

0 100 200 300 400 500

NO

2

- con

sum

ption

rate

(

mol g

-1 d

-1)

0.0

0.5

1.0

1.5

2.0

2.5

3.0+AB: R

2=0.65

Km=173 M NO2

-

Vmax=1.0 mol g-1

d-1

-AB: R2=0.70

Km=138 M NO2

-

Vmax=1.8 mol g-1

d-1

0 100 200 300 400 500

NO

2

- con

sum

ption

rate

(

mol g

-1 d

-1)

0

1

2

3

4

5+AB: R

2=0.75

Km=48 M NO2

-

Vmax=1.5 mol g-1

d-1

-AB: R2=0.85

Km=96 M NO2

-

Vmax=2.9 mol g-1

d-1

A

BNO2

- (M)

0 100 200 300 400 500

NO

2

- co

nsu

mp

tio

n r

ate

(

mo

l g

-1 d

-1)

0.0

0.5

1.0

1.5

2.0

2.5

3.0+AB: R

2=0.65

Km=173 M NO2

-

Vmax=1.0 mol g-1

d-1

-AB: R2=0.70

Km=138 M NO2

-

Vmax=1.8 mol g-1

d-1

0 100 200 300 400 500

NO

2

- co

nsu

mp

tio

n r

ate

(

mo

l g

-1 d

-1)

0

1

2

3

4

5+AB: R

2=0.75

Km=48 M NO2

-

Vmax=1.5 mol g-1

d-1

-AB: R2=0.85

Km=96 M NO2

-

Vmax=2.9 mol g-1

d-1

A

B

Spatial Scale: Nearest Neighbors and Niche Space

Raynaud & Nunan (2014) PLoS ONE 9:e87217

Conclusions about Nitrifier Niche Space

• It’s really more complicated than a single, explanatory variable, but generalities about drivers have been found • Numbers and activities of AOA do tend to dominate as:

• NH3 availability decreases

• pH decreases

• T increases

• Complexity of AOA and AOB responses to environmental drivers are a function of: • Genotypic and phenotypic variation among taxa

• Temporal variation in drivers

• Spatial heterogeneity

Acknowledgements

• Peter Bottomley

• Anne Taylor

• Xinda Lu

• Andrew Giguere

• Chris Burgess